A fundamental characteristic of malignant melanoma is resistance to apoptosis, which largely determines melanoma's resistance to therapy. The complex interaction of pro-apoptotic and anti-apoptotic members of the Bcl-2 family is a central control point of apoptosis and a promising drug target. The BH3 mimetic ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2/Bcl-XL/Bcl-w, and it exhibits promise for cancer treatment either as a single agent or in combination therapy. We have found that ABT-737 alone induced little cytotoxicity in melanoma cells, and knockdown experiments with RNAi techniques demonstrated that anti-apoptotic protein Mcl-1, but not Bcl-2 or Bcl-XL is the main mediator of melanoma resistance to ABT-737 treatment. In addition, ABT-737 displayed strong synergistic lethality when combined with either proteasome inhibitors (MG-132 or Bortezomib) or Paclitaxel, and mechanistic studies suggested that both proteasome inhibitors and Paclitaxel neutralize Mcl-1's functions. These exciting results indicate that drugs that neutralize Mcl-1 function are outstanding candidates for combination therapy with the ABT-737 for treating melanomas, and demand validation in preclinical models and further exploring the therapeutic potential of other drugs which target Mcl-1 in combination with ABT-737. It has also been proposed that cancer initiating cells (or cancer stem cells), which preferably initiate cancers, may be responsible for cancer resistance to therapy, and eliminating these cells may be essential for developing any cancer therapy with long term success. We will refer these cells as cancer imitating cells. We propose to investigate whether enriched melanoma initiating cells are more resistant to existing chemotherapeutic agents than non-sorted cells in melanoma, and to examine whether combinations of drugs that reduce multiple anti-apoptotic defenses such as ABT-737 plus Bortezomib or Paclitaxel will overcome this resistance of melanoma initiating cells. These studies will be an important component to understand whether melanoma initiating cells are more resistant to therapy. Specific Aim 1: To directly test the responses of enriched melanoma initiating cells to existing and new therapies in vitro. i) To determine whether enriched melanoma initiating cells are more resistant to conventional chemotherapeutic agents such as Temozolomide. ii) To determine whether combinations of ABT- 737 with either Bortezomib or Paclitaxel overcome the resistance in enriched melanoma initiating cells. Specific Aim 2: To verify the efficacy of the combinations of ABT-737 with either Bortezomib or Paclitaxel for treating melanoma using two novel xenotransplantation models: a model targeting human melanoma initiating cells in vivo, and a direct xenograft model for melanoma patient samples. Specific Aim 3: To investigate the therapeutic potential of combining BH3 mimetic ABT-737 with other compounds which inhibit Mcl-1. i) A new approach to down-regulating Mcl-1 in melanomas by microRNAs. ii) Other drugs known to inhibit Mcl-1. This project proposes to develop novel combination therapeutics by targeting multiple anti-apoptotic Bcl-2 members, and to verify the efficacy of the treatment combinations in killing melanomas, and most importantly, melanoma initiating cell populations. We believe that the studies proposed here will quickly lead to future clinical studies and to the clinical use of ABT-737 (or similar agents) in treating human melanoma patients. Importantly, this approach should not be restricted by different signaling signatures of individual melanomas. This proposal is a collaboration of experienced melanoma basic scientists, a skin stem cell expert, a melanoma oncologist, and an expert in xenotransplantation models of human cancers, and this team will make important advances in developing melanoma treatments based on reversing resistance to apoptosis.